The long term goal of this proposal is to understand molecular mechanisms that regulate microfilament reorganization during mitosis, as well as during oncogenic transformation of cultured cells. We will focus our studies on the biological significance of the mitosis-specific phosphorylation of caldesmon. We have demonstrated, for the first time, that nonmuscle caldesmon, a protein of relative molecular mass Mr = 83,000 which binds to actin and calmodulin, is disassociated from microfilaments during mitosis, apparently as a consequence of phosphorylation. We believe that this process may contribute to the changes of shape and structure seen in cells undergoing mitosis because caldesmon inhibits actomyosin ATPase, and because caldesmon, together with tropomyosin, regulates the actin severing and capping activities of gelsolin. We will biochemically characterize how the mitosis-specific phosphorylation of caldesmon changes microfilament assembly and actomyosin interactions in vitro. In vivo functions of mitosis-specific phosphorylation of caldesmon will be explored through microinjection of phosphorylated caldesmon, antibodies that block phosphorylation or dephosphorylation, and mutant caldesmons lacking the phosphorylation sites, into cultured cells. We will observe whether the microinjection will perturb the assembly of microfilaments during mitosis and other cell cycle stages. We will examine changes in the phosphorylation state of caldesmon during cell cycle progression in detail to see whether the phosphorylation of caldesmon correlates with changes in microfilament assembly. Because transformed cells show morphological alterations similar to those found in mitotic cells, we will examine, using ts Rat-I cells, whether caldesmon is phosphorylated during cell transformation. Finally, because we have found that cdc2 kinase (mitosis promotion factor) is one of two kinase activities that phosphorylate caldesmon during mitosis, we will characterize how cdc2 kinase and the other kinase activity phosphorylate caldesmon. We will also search for other microfilament-associated proteins that are phosphorylated by these kinases during mitosis. Because cell cycle control is intimately related to cell transformation, the proposed studies will help us understand not only how cells divide, but also why cancer cells lose control of cell division.